Lactoferrin is known as an iron-binding protein occurring in lacrima, saliva, peripheral blood, milk and the like. It is known that lactoferrin has antibacterial activity against coliform bacillus (Escherichia coli), staphylococcus and other enterobacteria (or enteric bacteria) in a concentration within the range of 0.5-30 mg/ml (Nonnecke, B. J. and Smith, K. L.; Journal of Dairy Science; Vol. 67, pp.606; 1984).
It has been considered in general that the antibacterial activity of lactoferrin is derived from the situation wherein environmental iron becomes unavailable to those microorganisms which require iron strongly, due to the chelation of lactoferrin with environmental iron. The antibacterial activity of lactoferrin is not necessarily strong enough, thus a considerable quantity of lactoferrin is required to utilize its antibacterial activity, especially when lactoferrin is added to, impregnated into, adhered to, or coated onto other materials. Thus, there is a limitation of its usefulness as an antibacterial agent.
It has been attempted to increase the antibacterial activity of lactoferrin. For example, it has been proposed to use lactoferrin together with lysozyme (Japanese Unexamined Patent Application Gazette No. 62(1987)-249931). It has been also reported that the copresence of lactoferrin and secretory IgA may multiplicatively augment antibacterial activity of the former (S. Stephens, J. M. Dolby, J. Montreuil and G. Spik; Immunology; Vol. 41; Page 597; 1980).
To the best knowledge of the inventors, however, there has been no report indicating that chemical treatment of lactoferrin may improve its antibacterial activity.
It is also known that lactoferrin is unstable to heating, and that the antibacterial activity of lactoferrin can be almost completely suppressed by heating it at 62.5.degree. C. for 30 minutes, and complete deactivation is achieved by heating it at 70.degree. C. for 15 minutes (Ford, J. E. et al; Journal of Pediatrics, Vol. 90, page 29; 1977).
Therefore, sufficient thermal treatment cannot be applied to those materials which contain lactoferrin as an antibacterial agent.
Also, it is known that lactoferrin is not stable to pH variation.
The inventors of the present invention have exerted their efforts to increase the antibacterial activity of lactoferrin and to improve its stability to heating, and found that hydrolyzates of lactoferrin substances such as native lactoferrin, apolactoferrin, metal saturated lactoferrin, and mixtures thereof show much stronger antibacterial activity and superior stability to heating than unhydrolyzed lactoferrin. The present invention is based on this discovery. The words "native lactoferrin" used herein means that lactoferrin was just isolated from milk and the like, and that no chemical treatment such as iron removing and chelation with metals is made thereon.
Meanwhile it has not been known that lactoferrin and its hydrolyzates have potent tyrosinase inhibition activity.
Tyrosinase is known as an enzyme which may act as a catalyst for the oxidization of tyrosine, and other monohydric phenols and corresponding dihydric orthophenols with molecular oxygen. Tyrosinase widely occurs in plants such as mushrooms, potatoes, apples as well as in animal tissues. It is also known that tyrosinase is related to darkening phenomena at an injured portion of plant tissue, and is also related to the formation of melanin pigment in various tissues of animals, especially in epidermal cells (Editorial Committee of Encyclopedia Chimica, Encyclopedia Chimica, Vol. 5, page 976, Kyohritsu Shuppan; 1960).
It is also known that pigmentation of melanin in epidermal cells or mucous membranes in Addison's disease results from a decrease in secretion of adrenal cortex hormones which antagonize melanotropin which in turn promotes tyrosinase activity (Editorial Committee of Encyclopedia Chimica, Encyclopedia Chimica, Vol. 1, Page 65, Kyohritsu Shuppan; 1960).
Therefore, it has been strongly desired, in the industrial fields of pharmaceuticals, cosmetics, food and the like, to develop a tyrosinase inhibition agent for prevention and therapy of symptoms resulting from undesirable effects of tyrosinase activity. Especially in the cosmetics industry, research has been actively made on cosmetics or medicines for external use for effective inhibition of melanin-formation and for whitening of skin, and many products containing tyrosinase inhibition agents have been successively developed. There are known many tyrosinase inhibition agents, for example, cysteine and vitamin C (Yutaka Mishima et al, Fundamental Dermatology, page 258, Asakura Shoten; 1973), kojic acid (Nikkei Sangyo Newspaper, May 24th 1988), arbutin (Ken-ichi Tomita, Preliminary Text for 20th F. J. Seminar, page 21, Fragrance Journal Company, Mar. 14, 1990), products of microorganism belonging to the genus of Trichoderma (Unexamined Japanese Patent Application Gazette No. 2(1990)-145189).
The conventional tyrosinase inhibition agents, however, had more or less defects in that they are unstable in the products, they are excessively potent to melanocytes which produce melanin pigment, they are too expensive due to the difficulty in obtaining their raw materials, and they are not usable as cosmetics or medicines for external use from the view points of safety, economics, preservability, reliability for whitening effect and so on.
The inventors of the present invention found that lactoferrin, especially its hydrolyzate, has potent tyrosinase-inhibition activity. The present invention is also based on this discovery.